PROJECT SUMMARY/ABSTRACT Cholangiopathies are incurable, progressive extrahepatic bile duct (EHBD) disorders characterized by injury- induced cholangiocyte hyperproliferation. Development of therapies for cholangiopathies requires better understanding of cellular and molecular mechanisms regulating EHBD proliferation during homeostasis and in response to injury. Molecular pathways that regulate cholangiocyte proliferation are currently not well understood. This application aims to answer a fundamental question about mechanisms of EHBD regeneration and explore strategies to modulate the proliferative potential of the biliary epithelium. The rationale for the proposed research is that defining cellular and molecular interactions underlying cholangiocyte responses to injury can offer novel therapeutic strategies for cholangiopathies and hepatobiliary regenerative medicine. Our preliminary data suggest that Hedgehog (HH) and WNT signaling play important roles in EHBD homeostasis and proliferation after injury. The overarching hypothesis for this proposal is that HH and WNT signaling regulate crosstalk between epithelial and stromal cells to promote EHBD proliferation after injury. In this proposal, we will use genetic mouse models and pharmacological treatments in vivo. Bile duct ligation will be used as an injury model. Human and mouse EHBD organoid (BDO) co-culture with primary mesenchymal cells will be used to directly study epithelial-stromal crosstalk and pathway interaction in vitro and enhance the translational component of this project. Aim 1 will focus on HH signaling and determine if the Indian HH ligand from cholangiocytes signals to GLI1+ HH-responsive fibroblasts to indirectly regulate cholangiocyte proliferation. Aim 2 will focus on WNT signaling and test if WNT from GLI1-expresing cells is critical for cholangiocyte proliferation. Aim 3 will focus on cell-cell and pathway interactions and determine if IHH from cholangiocytes directly regulates WNT production by fibroblasts to induce cholangiocyte proliferation. Under Aims 1 and 2 we will use transgenic reporter and loss-of-function mouse models, pharmacological inhibitors of HH and WNT signaling, in situ hybridization, and immunohistochemistry techniques to define the in vivo effects. Under Aim 3 we take a reductionist approach by using organoid co-culture models. It will also support the development of robust new tools (transgenic mice and organoid models) and skills (in situ hybridization and flow cytometry) to support an independent research program focused on fundamental signaling pathways regulating EHBD. This new K08 application will also promote the development of the PI into an independent NIH-funded investigator and support her long-term goal to understand fundamental mechanisms of EHBD biology to ultimately improve outcomes in patients with cholangiopathies.